Research on the mechanisms of action of the renin-angiotensin system has provided a wealth of information about its role in both physiology and cardiovascular pathology. Characterization of newer biochemical pathways by which angiotensin peptides act on diverse cellular receptors to exert modulatory functions through paracrine and intracrine mechanisms include the identification of angiotensin converting enzyme 2 (ACE2), the vasodilator and antiproliferative peptide angiotensin-(1-7) [Ang-(1-7)], and the mas-receptor. Upstream within the biochemical cascade leading to the formation of angiotensin peptides, angiotensinogen (Aogen) remains the undisputed large molecular protein from which Ang II and Ang-(1-7) are generated. This application details studies directed to determine whether a newly described propeptide, cleaved from Aogen by a non-renin pathway, may be an alternate substrate for the formation of the biologically active angiotensins. The dodecapeptide angiotensin-(1-12) [Ang-(1-12)], isolated from rat tissues, has been found to generate Ang II and Ang-(1-7) by a non-renin enzyme and be selectively expressed in large quantities in cardiac myocytes of spontaneously hypertensive rats (SHR). The major goal of the proposed studies is to identify the mechanisms regulating the expression and function of Ang-(1-12) in the heart of both normotensive Wistar Kyoto (WKY) and SHR, explore the intracellular and extracellular pathways by which the substrate is cleaved from Aogen, and assess whether Ang-(1-12) is an alternate or obligatory intermediate peptide for the intracellular action of Ang II and Ang-(1-7). These objectives will be achieved by a combination of histochemical, molecular, cellular, and physiological experiments using both in vivo and in vitro approaches. The research plan includes four Specific Aims. Specific Aim \ will determine the expression of Ang-(1-12) in the cellular components of the heart tissue of normal WKY and SHR using a combination of quantitative immunohistochemistry (IHC) and confocal microscopy. Specific Aim 2 will assess the metabolic pathways for Ang-(1-12) formation and processing into Ang I, Ang II, and Ang-(1-7) in the heart of both strains. In Specific Aim 3 we will evaluate whether Ang-(1-12) is incorporated or synthesized by cardiac myocytes in culture and whether the presence of the alternate substrate activates a signaling pathway mimicking the effects of Ang II. Specific Aim 4 will evaluate the role of Ang-(1-12) in the regulation of arterial pressure and cardiac function in both WKY and SHR through assessing the effect of graded doses of Ang-(1-12) infusions, measuring endogenous inhibition of Ang-(1-12) through the administration of a polyclonal Ang-(1-12) antibody to sequester the peptide, or determining the effects of chronic inhibition of renin and other Ang I- and Ang ll-forming enzymes on plasma and cardiac tissue levels of Ang-(1-12). These studies will allow identification of alternate pathways for angiotensin peptide formation upstream of angiotensin I (Ang I), discoveries that should have a major impact in understanding the relative efficacy of renin inhibitors in cardiac remodeling and the development of newer agents that would block the enzyme(s) by which Ang-(1-12) is processed to the active angiotensin peptides.